Abstract：
More and more ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae have been reported with its widespread use, and the detection rate of KPC variants has increased dramatically. However, the evolutionary mechanism and fitness effects during KPC mutation remained unknown. Here, we report the complex in vivo evolutionary trajectories of two novel KPC variants, KPC-155 (L169P/GT242A) and KPC-185 (D179Y/GT242A), from K. pneumoniae in the same patient. The novel variants were shown to confer ceftazidime-avibactam resistance but restore carbapenem susceptibility based on the results of plasmid transformation assays, cloning experiments, and enzyme kinetic measurements. In vitro, competition experiments highlighted the adaptive advantage conferred by strains carrying these KPC variants, which could lead to the rapid spread of these ceftazidime-avibactam-resistant strains. The growth curve indicated that blaKPC-185 had better growth conditions at lower avibactam concentration compared to blaKPC-155, which was consistent with ceftazidime-avibactam use in vivo. In addition, replicative transposition of the IS26-flanked translocatable unit (IS26-ISKpn6-blaKPC-ISKpn27-IS26) also contributes to the blaKPC amplification and formation of two copies (blaKPC-2 and blaKPC-185), conferring both carbapenem and ceftazidime-avibactam resistance. However, strains with double copies showed reduced competitive advantage and configuration stability. The comparative plasmid analysis of IS26 group (IS26-blaKPC-IS26) and Tn1721 group (Tn1721-blaKPC-IS26) revealed that IS26-insertion could influence the distribution of resistance genes and ability of self-conjugation. The dynamic changes in blaKPC configuration highlight the need for consistent monitoring including antimicrobial susceptibility testing and determination of blaKPC subtypes - during clinical treatment, especially when ceftazidime-avibactam is administered.
摘要：
越来越多的头孢他啶-阿维巴坦耐药的产KPC肺炎克雷伯菌被报道,KPC变异体的检出率显著提高。然而,KPC突变过程中的进化机制和适应性效应尚不清楚.这里,我们报告了两个新的KPC变体的复杂体内进化轨迹，KPC-155(L169P/GT242A)和KPC-185(D179Y/GT242A),来自同一患者的肺炎克雷伯菌。根据质粒转化试验的结果，新的变体被证明赋予头孢他啶-阿维巴坦抗性，但恢复碳青霉烯敏感性。克隆实验,和酶动力学测量。体外竞争实验强调了携带这些KPC变体的菌株赋予的适应性优势，这可能导致这些头孢他啶-阿维巴坦耐药菌株的快速传播。生长曲线表明,与blaKPC-155相比,blaKPC-185在较低的阿维巴坦浓度下具有更好的生长条件,这与在体内使用头孢他啶-阿维巴坦一致。此外,IS26侧翼转位单元（IS26-ISKpn6-blaKPC-ISKpn27-IS26）的复制转座也有助于blaKPC扩增和形成两个拷贝（blaKPC-2和blaKPC-185），同时赋予碳青霉烯类和头孢他啶-阿维巴坦抗性。然而,具有双拷贝的菌株显示出降低的竞争优势和构型稳定性。IS26组（IS26-blaKPC-IS26）和Tn1721组（Tn1721-blaKPC-IS26）的比较质粒分析显示，IS26插入可能会影响抗性基因的分布和自结合能力。blaKPC构型的动态变化突出表明需要在临床治疗期间进行一致的监测，包括抗菌药物敏感性测试和确定blaKPC亚型。尤其是服用头孢他啶-阿维巴坦时。
